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. 1988 Aug 1;107(2):651–664. doi: 10.1083/jcb.107.2.651

Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells

PMCID: PMC2115201  PMID: 3047145

Abstract

To study microtubule (MT) dynamics in nerve cells, we microinjected biotin-labeled tubulin into the cell body of chemically fused and differentiated PC12 cells and performed the immunofluorescence or immunogold procedure using an anti-biotin antibody followed by secondary antibodies coupled to fluorescent dye or colloidal gold. Incorporation of labeled subunits into the cytoskeleton of neurites was observed within minutes after microinjection. Serial electron microscopic reconstruction revealed that existing MTs in PC12 neurites incorporated labeled subunits mainly at their distal ends and the elongation rate of labeled segments was estimated to be less than 0.3 micron/min. Overall organization of MTs in the nerve cells was different from that in undifferentiated cells such as fibroblasts. Namely, we have not identified any MT-organizing centers from which labeled MTs are emanating in the cell bodies of the injected cells. Stereo electron microscopy revealed that some fully labeled segments seemed to start in the close vicinity of electron dense material within the neurites. This suggests new nucleation off some structures in the neurites. We have also studied the overall pattern of the incorporation of labeled subunits which extended progressively from the proximal part of the neurites toward their tips. To characterize the mechanism of tubulin incorporation, we have measured mean density of gold labeling per unit length of labeled segments at different parts of the neurites. The results indicate access of free tubulin subunits into the neurites and local incorporation into the neurite cytoskeleton. Our results lead to the conclusion that MTs are not static polymers but dynamic structures that continue to elongate even within the differentiated nerve cell processes.

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Selected References

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  1. Baas P. W., White L. A., Heidemann S. R. Microtubule polarity reversal accompanies regrowth of amputated neurites. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5272–5276. doi: 10.1073/pnas.84.15.5272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Black M. M., Keyser P., Sobel E. Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J Neurosci. 1986 Apr;6(4):1004–1012. doi: 10.1523/JNEUROSCI.06-04-01004.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brady S. T., Tytell M., Lasek R. J. Axonal tubulin and axonal microtubules: biochemical evidence for cold stability. J Cell Biol. 1984 Nov;99(5):1716–1724. doi: 10.1083/jcb.99.5.1716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bray D., Bunge M. B. Serial analysis of microtubules in cultured rat sensory axons. J Neurocytol. 1981 Aug;10(4):589–605. doi: 10.1007/BF01262592. [DOI] [PubMed] [Google Scholar]
  5. Graessmann A., Graessmann M., Mueller C. Microinjection of early SV40 DNA fragments and T antigen. Methods Enzymol. 1980;65(1):816–825. doi: 10.1016/s0076-6879(80)65076-9. [DOI] [PubMed] [Google Scholar]
  6. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hatanaka H. Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line. Brain Res. 1981 Oct 19;222(2):225–233. doi: 10.1016/0006-8993(81)91029-5. [DOI] [PubMed] [Google Scholar]
  8. Heidemann S. R., Hamborg M. A., Thomas S. J., Song B., Lindley S., Chu D. Spatial organization of axonal microtubules. J Cell Biol. 1984 Oct;99(4 Pt 1):1289–1295. doi: 10.1083/jcb.99.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heidemann S. R., Landers J. M., Hamborg M. A. Polarity orientation of axonal microtubules. J Cell Biol. 1981 Dec;91(3 Pt 1):661–665. doi: 10.1083/jcb.91.3.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hiller G., Weber K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell. 1978 Aug;14(4):795–804. doi: 10.1016/0092-8674(78)90335-5. [DOI] [PubMed] [Google Scholar]
  11. Hirokawa N. 270K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon. J Cell Biol. 1986 Jul;103(1):33–39. doi: 10.1083/jcb.103.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirokawa N., Bloom G. S., Vallee R. B. Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta'-iminodipropionitrile-intoxicated axon as a model system. J Cell Biol. 1985 Jul;101(1):227–239. doi: 10.1083/jcb.101.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirokawa N., Glicksman M. A., Willard M. B. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J Cell Biol. 1984 Apr;98(4):1523–1536. doi: 10.1083/jcb.98.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoffman P. N., Lasek R. J. The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J Cell Biol. 1975 Aug;66(2):351–366. doi: 10.1083/jcb.66.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jacobs J. R., Stevens J. K. Changes in the organization of the neuritic cytoskeleton during nerve growth factor-activated differentiation of PC12 cells: a serial electron microscopic study of the development and control of neurite shape. J Cell Biol. 1986 Sep;103(3):895–906. doi: 10.1083/jcb.103.3.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Joshi H. C., Baas P., Chu D. T., Heidemann S. R. The cytoskeleton of neurites after microtubule depolymerization. Exp Cell Res. 1986 Mar;163(1):233–245. doi: 10.1016/0014-4827(86)90576-8. [DOI] [PubMed] [Google Scholar]
  18. Kristofferson D., Mitchison T., Kirschner M. Direct observation of steady-state microtubule dynamics. J Cell Biol. 1986 Mar;102(3):1007–1019. doi: 10.1083/jcb.102.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Letourneau P. C. Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J Cell Biol. 1983 Oct;97(4):963–973. doi: 10.1083/jcb.97.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mitchison T., Evans L., Schulze E., Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. doi: 10.1016/0092-8674(86)90283-7. [DOI] [PubMed] [Google Scholar]
  21. O'Lague P. H., Huttner S. L. Physiological and morphological studies of rat pheochromocytoma cells (PC12) chemically fused and grown in culture. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1701–1705. doi: 10.1073/pnas.77.3.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richter-Landsberg C., Jastorff B. The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells. J Cell Biol. 1986 Mar;102(3):821–829. doi: 10.1083/jcb.102.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salmon E. D., Leslie R. J., Saxton W. M., Karow M. L., McIntosh J. R. Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching. J Cell Biol. 1984 Dec;99(6):2165–2174. doi: 10.1083/jcb.99.6.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Salmon E. D., Saxton W. M., Leslie R. J., Karow M. L., McIntosh J. R. Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching. J Cell Biol. 1984 Dec;99(6):2157–2164. doi: 10.1083/jcb.99.6.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sammak P. J., Gorbsky G. J., Borisy G. G. Microtubule dynamics in vivo: a test of mechanisms of turnover. J Cell Biol. 1987 Mar;104(3):395–405. doi: 10.1083/jcb.104.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schulze E., Kirschner M. Dynamic and stable populations of microtubules in cells. J Cell Biol. 1987 Feb;104(2):277–288. doi: 10.1083/jcb.104.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shiomura Y., Hirokawa N. Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy. J Cell Biol. 1987 Jun;104(6):1575–1578. doi: 10.1083/jcb.104.6.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shiomura Y., Hirokawa N. The molecular structure of microtubule-associated protein 1A (MAP1A) in vivo and in vitro. An immunoelectron microscopy and quick-freeze, deep-etch study. J Neurosci. 1987 May;7(5):1461–1469. doi: 10.1523/JNEUROSCI.07-05-01461.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Soltys B. J., Borisy G. G. Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes. J Cell Biol. 1985 May;100(5):1682–1689. doi: 10.1083/jcb.100.5.1682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thompson W. C., Asai D. J., Carney D. H. Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin. J Cell Biol. 1984 Mar;98(3):1017–1025. doi: 10.1083/jcb.98.3.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yamada K. M., Spooner B. S., Wessells N. K. Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol. 1971 Jun;49(3):614–635. doi: 10.1083/jcb.49.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]

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